Human infection with influenza virus poses a significant annual global health threat, especially when they are complicated by secondary bacterial infections. During the recent H1N1 pandemic, S. pyogenes has caused about 1/3 of the deaths associated with bacterial secondary infections; however, relatively little is known about influenza-S. pyogenes superinfections. Influenza infection induces thrombosis, which results in fibrinogen/fibrin (Fb) and fibronectin (Fn) deposition in the respiratory tract. These ae well-known ligands involved in S. pyogenes adherence and aggregation and its ability to resist killing by macrophages. We hypothesize that influenza-induced thrombosis promotes superinfections due, in part, to the binding of Fb and Fn to the surface of the pathogen, which increases bacterial adherence and biofilm formation, and by diminishes immune clearance. To test this hypothesis, we will complete the following specific aims: 1) Delete each of the two Fb/Fn binding proteins (PrtF.2 and M3) of S. pyogenes strain MGAS315 and determine the contribution of each to Fb and Fn binding. 2) Determine if PrtF.2 and M3 proteins influence adherence to epithelial cells infected with influenza, and biofilm formation, in a Fb/Fn-dependent manner. 3) Determine if PrtF.2 and M3-mediated binding of Fb/Fn alters the efficiency of macrophage killing of S. pyogenes either with, or without, antecedent influenza exposure. 4) Determine if PrtF.2 and M3 protein contribute to the excess morbidity and mortality in an established murine model. The project addresses an important public health concern and may lead to new ways to mitigate the excess morality associated with influenza. PUBLIC HEALTH RELEVANCE: Influenza causes an average of 250-500,000 hospitalizations and 36,000 deaths on an annual basis in the U.S. Death is typically due to the development of secondary bacterial complications, which is called excess mortality, or superinfection. During the recent flu pandemic, Streptococcus pyogenes caused about 1/3 of the known cases of superinfection. The factors that make people susceptible to superinfection are poorly understood; however, influenza infection was recently discovered to cause thrombosis resulting the deposition of fibrinogen (Fb) and fibronection (Fn) in the respiratory tract. Fb and Fn binding to the surface of S. pyogenes via specific receptors enhances pathogen adherence to human tissue and confers resistance to the innate immune system. The project will determine the role of Fb/Fn binding to bacteria as a cause of superinfections. The results will be important in assessing, and mitigating with new treatment approaches, the morbidity and mortality associated with the flu.